Mutagenesis and molecular dynamics suggest structural and functional roles for residues in the N-terminal portion of the cytochrome P450 2B1 I helix.

Emily E Scott, Hong Liu, You Qun He, Weihua Li, James R Halpert

Index: Arch. Biochem. Biophys. 423(2) , 266-76, (2004)

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Abstract

To investigate their potential roles in ligand access, binding, and subsequent metabolism, residues in the N-terminal portion of the cytochrome P450 2B1 I helix were mutated to alanine and phenylalanine. Of the 18 mutants from E286 to S294 only 7 yielded holoprotein in an Escherichia coli expression system. Substitutions at positions 289, 290, 292, and 294 caused >/= 2-fold changes in kcat and/or Km for two or more of the 2B1 substrates examined, testosterone, 7-ethoxy-4-trifluoromethylcoumarin, 7-benzyloxyresorufin, and benzphetamine. I290 substitutions had the largest effects on steady-state parameters for three substrates and increased benzphetamine affinity. Steered molecular dynamics simulations of testosterone egress along the I helix identified hydrophobic interactions with I290, L293, and S294 and water bridges to E286 and S294. Sensitivity of holoprotein formation to substitution and effects on substrate binding and metabolism suggest structural and functional roles for residues in the N-terminus of the cytochrome P450 2B1 I helix.